鸡骨胶原蛋白肽增强肠道免疫活性的构效机制研究

Bone residue is the byproduct of meat industry rich in collagen, which is both abundant and readily available but greatly underutilized as a protein source. It is thus critical to develop an effective way to use this valuable protein source. Type I collagen is the main collagen of chicken hard bone, while few reported studies were found in utilization of it due to lacking of efficient ways to extract it out. The immuno-regulatory peptides from hydrolyzed collagen I extracted with hot-pressure extraction from chicken bone residue will be purified with series chromatography including Sepdex G-25 gel filtration, Ion exchange chromatography, HPLC in the present study. Their immunomodulating ability will be evaluated in lymphocyte proliferation assay, macrophage phagocytosis capacity and DNA protection model, and thus Chicken Bone Collagen Peptides (CCP) with higher immunomodulating effects will be obtained, and the amino acids sequence of CCP will be characterized through LC-MS-MS determination and ExPASy data analysis. Composition of CCP obtained at different stage of purification will be determined and their dose-dependent immunomodulating capacity in both cell line and SD rats will be tested as the in vitro and in vivo effects, to demonstrate the correlation of CCP among the structure, composition, dose and immunomodulating effect through determination of DNA protection effect, expression of IgG, IgA, cytokines and histological section of small intestine. The present study is aiming to establish a foundation elucidating the immunomodulating pathway of collagen peptides derived from chicken bone residue, and thus provide theoretical parameters on how to change animal bone residue as a byproduct into value-added products.

畜禽骨作为肉品加工业的副产物，利用率低，资源浪费严重。鸡骨富含胶原蛋白，其中硬质骨中所含I型胶原蛋白为其主要亚型，但关于其研究和利用因缺乏有效制备方法而受到制约。本项目以鸡骨副产物为原料，采用热压抽提耦连靶向酶解的方法，高效提取制备鸡骨胶原蛋白肽。借助DNA损伤保护作用、腹腔巨噬细胞吞噬作用和脾细胞增值作用等体外评价方法，筛选免疫调节活性高的肽段，进行多步色谱联合分离纯化，采用LC-MS-MS测定结合ExPASy数据库分析，鉴定鸡骨胶原蛋白肽的氨基酸序列；通过对不同纯度、组成与剂量的肽段的细胞免疫活性测定和大鼠的肠道免疫调节效应验证，借助免疫因子体系的特征表达，在细胞和生物体水平探明摄入胶原蛋白肽的组成、剂量与其免疫调节作用的关联效应，阐明鸡骨胶原蛋白肽增强肠道免疫活性的构效机制。本项目为探讨骨源多肽调节机体免疫活性的作用机制奠定基础，并为畜禽骨高值化利用提供理论依据。

本项目以鸡骨为原料，采用酸法和酶法提取鸡骨胶原蛋白（肽）并对其进行结构鉴定，明确了酶溶胶原蛋白（肽）提取率、蛋白含量、羟脯氨酸含量和变性温度均高于酸溶胶原蛋白（肽），由α链（α1和α2）、β链和γ链组成，具有 I型胶原蛋白（肽）的组成特征，较好的保留了胶原蛋白三股螺旋结构；探讨了不同加热温度和时间对鸡骨胶原蛋白（肽）结构和凝胶特性的影响机制，明确了随着加热进行，蛋白溶解度显著升高，水解度显著降低，蛋白（肽）发生降解，同时小分子蛋白发生交联聚合，胶原蛋白二级结构改变，β-折叠相对百分含量显著下降，无规则卷曲显著上升，90℃加热胶原蛋白得到的水解物制备的凝胶特性最优，该结果为鸡骨的综合利用提供了理论与技术的支撑。